| Objective (1) To establish a rat glaucoma model. (2)To observe the change of cellular localization of ciliary neurotrophic factor in retina of rat glaucoma model compare with normal rat.(3) To study the effects of ciliary neurotrophic factor on cytoactive and processes regrowth of purified rat retinal ganglion cells in vitro. Methods (1) The rat glaucoma model with chronic increased intraocular pressure which was induced by electrocoagulation of the episcleral and limbal veins. (2) The retinas were harvested in 1st day, 7th day and 28th day after the model was established. Immunohistochemical staining was used to localize the cells those express ciliary neurotrophic factor. (3) SD rat (1～3 days after birth) retinas were dissected and dissociated. Retinal ganglion cells were purified by grit and cultured in 96-wells plates. 5 sorts of culture medium which separately contain 0ng·ml-1,10ng·ml-1,20ng·ml-1,30ng·ml-1,40ng·ml-1 ciliary neurotrophic factor were prepared. 24 hours after cells were seeded, 4 sorts of medium which contain ciliary neurotrophic factor were added in, and Retinal ganglion cells cultured without ciliary neurotrophic factor were used as control. The cells were identified by morphologic criteria and immunocytochemical staining. In 1st d, 3rd d, 5th d, 7th d, 9th d, 11th d, 13th d, 15th d after ciliary neurotrophic factor was added, MTT colorimetric assay was adopted to determine the cytoactive of cultured retinal ganglion cells cultured with different concentration of ciliary neurotrophic factor. The numbers of RGCs and cells with processes in every random microscopic fields were determined [400x magnification using fluorescence microscopy and phase-contrast microscopy]. Rusults (1) The rat glaucoma model was successfully established. (2) In normal rat retina, ciliary neurotrophic factor was mainly localized in ganglion cell layer. In rat glaucoma model retina, ciliary neurotrophic factor was localized in ganglion cell layer, outer nuclear layer and inner nuclear layer. (3) Between 1st day and 7th day when ciliary neurotrophic factor was added, the cytoactive of cells cultured with higher concentration of ciliary neurotrophic factor was higher. In 7th day, cytoactive of every two groups are significantly different(p<0.05). Between 13th day and 15th day when ciliary neurotrophic factor was added, the enhancing effect of 40 ng·ml-1 ciliary neurotrophic factor on cytoactive was in a decrease trend compared with 20ng·ml-1 and 30 ng·ml-1 ciliary neurotrophic factor(p<0.05). Between 3rd day and 5th day when CNTF was added, the process rate of RGCs cultured with 40ng·ml-1 CNTF was significantly higher than control (P<0.05).Between 7th day and 15th day ,the process rates of RGCs cultured with 20ng·ml-1, 30ng·ml-1, 40ng·ml-1 CNTF were significantly higher than control(P<0.05), and the process rates of RGCs cultured with 30ng·ml-1, 40ng·ml-1 CNTF were significantly higher than RGCs cultured with 10ng·ml-1, 20ng·ml-1 CNTF(P<0.05).Conclusion (1) Rat glaucoma model can be established applying previous described medthod.(2) Ciliary neurotrophic factor was up-regulated in rat glaucoma model. (3) Ciliary neurotrophic factor can significantly enhancing the cytoactive of purified cultured retinal ganglion cells, and a dose dependent manner can be determined between 1st day and 7th day. Between 13th day and 15th day when ciliary neurotrophic factor was added, the enhancing effect of 40 ng·ml-1 ciliary neurotrophic factor on cytoactive was in a decrease trend. CNTF can significantly enhance processes regrowth of purified rat RGCs in vitro, and a dose-dependent relationship has been determined. In the definite concentration domain, higher concentration of CNTF can enhance processes regrowth earlier. |
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